The continuing long-term goal of research conducted under R01 DE015931 is to identify the in vivo conditions of the gingival sulcus that promote the growth and virulence of P. gingivalis, so triggering periodontitis. The hypothesis is that the role of P. gingivalis in this host-pathogen interaction is manifested through its responses to environmental conditions that prevail in the subgingival crevice. Like other bacteria, in P. gingivalis these responses are regulated by two-component signal transduction systems. In the simplest system the first component, a histidine kinase (HK), responds to a specific environmental stimulus by auto-phosphorylating a designated histidine residue in its sequence. Information is relayed by transfer of activated phosphate from the HK histidine to a designated aspartate residue in the cognate response regulator (RR). The activated RR then binds to promoters of its target genes (the regulon) and regulates their expression. In Specific Aim 1 of this application the regulons of P. gingivalis RRs will be identified by ChIP-on-chip and complementary assays. In Specific Aim 2, direct interaction between RRs and promoters of target genes will be confirmed by gel shift assays, and target promoter sequences will be analyzed. There is relatively limited information on the inducers of two-component systems compared to that available on the molecular mechanisms of gene regulation. A goal of this application is to fill this knowledge gap and identify environmental conditions to which P. gingivalis RRs respond. At this point in the project validated information on each RR and its regulon will be used to make informed decisions on potential conditions that trigger activation. Therefore, in Specific Aim 3 the environmental cues that activate individual HK-RR systems will be identified using reporter gene constructs. It is claimed that histidine kinases and response regulators are good therapeutic targets because of their multiple roles in bacterial physiology. Though the development of specific inhibitors has been slow, small molecule inhibitors for bacterial transcriptional regulators were recently discovered in high through-put screens of chemical libraries, reviving interest in these potential targets. In the future, such inhibitors may have a positive impact on general human health and influence treatments for periodontal disease replacing less effective and riskier antibiotic therapies. PROJECT NARRATIVE: The goal of this research is to identify conditions within the gingival pocket that promote the growth of bacteria that cause periodontitis. Bacterial responses to these conditions are regulated by signal relay systems comprised of two components: histidine kinases that sense changes in the environment and regulators that co- ordinate the response to these changes. Such two-component systems are prime targets for new inhibitor therapeutics that positively impact human health.